Project Summary One third of the world's population carries the infectious agent Mycobacterium tuberculosis (Mtb) that causes tuberculosis (TB). Current treatments for TB disease are not straightforward. Drug resistance to TB drugs results from insufficient treatments that select for resistance, as well as from inherently resistant popula- tions. Because of the arduous and difficult to follow regimen for TB treatment, there were approximately 480,000 cases of multi-drug resistant TB (MDR-TB) and 100,000 cases of rifampicin-resistant TB (RR-TB) in 2015. Treatment of MDR-TB has a 52% success rate, and about 15% of cases develop into extensively-drug resistant TB (XDR-TB), which has been found in 117 countries. Multi-drug resistant TB requires treatment for two years with a cocktail of at least 5 drugs. New drug with mechanisms of action that eradicate persistence and drug tolerant populations will reduce treatment times and the spread of virulent drug resistant strains. We propose that Mtb cholesterol metabolism contributes to persis- tence in the host and presents a target for therapeutics with new mechanisms of action. Our studies will pro- vide much needed information about mechanism of oxidative stress resistance in Mtb and how these mecha- nisms are tied to cholesterol metabolism. Upon completion, (1) we will identify the molecular target of a TB drug potentiator that has the capacity to shorten TB treatment times. (2) We will characterize the biochemical function of a regulon that is only encoded in mycobacterial pathogens, and which our preliminary data suggest contains the target of our potentiators. (3) We will model control of metabolite flow between cholesterol catabo- lism and ROS resistance pathways. Taken together, these studies will identify vulnerable targets for drug dis- covery that eliminates Mtb persistence and drug tolerance.